Chaitali S P Tendulkar, PES RTBCOP 1

SYLLABUS
• High performance liquid chromatography: -
Introduction,
theory,
instrumentation,
advantages and applications.

Chaitali S P Tendulkar, PES RTBCOP 2

LIQUID CHROMATOGRAPHY
• Liquid chromatography in which the mobile phase is a liquid
• The liquid used as the mobile phase is called eluant
• Stationary phase may be solid or liquid
• Separation occurs based on the interaction of the sample which
stationary and mobile phase
• Liquid column chromatography-----
a. Classical column open chromatography
b. High Performance Liquid chromatography

Chaitali S P Tendulkar, PES RTBCOP 3

HPLC
• High Performance Liquid chromatography
• Type of closed column chromatography which requires use of high
pressure to pump the mobile phase through the column
• In the beginning– pumps only had a pressure capability of 500psi
(High Pressure Liquid Chromatography)
• New HPLC instruments could develop pressure upto 6000psi,
incorporated new injectors, detectors and columns– advanced
performance (smaller particle, greater performance)– renamed High
Performance Liquid Chromatography

Chaitali S P Tendulkar, PES RTBCOP 4

DIASADVANTAGES OF CLASSICAL COLUMN
CHROMATOGRAPHY
• Column packing procedures are tedious, columns usually
used only once– makes the technique expensive
• Efficiency achieved by these large packing particles in
columns is relatively low and analysis time is lengthy
• Detection of solute in eluant is achieved by manual analysis
of individual sections which is laborious and time-consuming

TLC offered an improvement in some aspects though it suffered from

limitations such as automation, reproducibility, quantitation, and
preparative studies
Chaitali S P Tendulkar, PES RTBCOP 5
• GAS CHROMATOGRAPHY
• Overcame most drawbacks of Cassical Column Chromatography and TLC
üAutomation possible
üBetter quantitation
üBetter reproducibility
üColumn efficiency is better

⨯Sample has to be volatile (has to be gas or derivatized to a gas)

⨯Most detectors are destructive of sample

Chaitali S P Tendulkar, PES RTBCOP 6

ADVANTAGES OF HPLC
üMethod of choice for non-volatile, thermolabile samples
üSuperior quantitative capability and reproducibility
üHigh separation capacity, enabling the batch analysis of multiple
components
üModerate analytical conditions
ØUnlike GC, the sample does not need to be vaporised
üGenerally high sensitivity
üLow sample consumption
üEasy preparation of separation and purification of samples
Chaitali S P Tendulkar, PES RTBCOP 7
INSTRUMENTATION
• Solvent reservoir and solvent
treatment system
• Pumps (Reciprocating,
displacement, pneumatic)
• Sample injection system
• Mobile phase
• Columns, column packings,
Stationary phase
• Fittings (for normal and reverse
phase, analytical and guard
column, thermostat)
• Detectors
Chaitali S P Tendulkar, PES RTBCOP 8
Chaitali S P Tendulkar, PES RTBCOP 9
INSTRUMENTATION

Chaitali S P Tendulkar, PES RTBCOP 10

WORKING OF HPLC
• Filtered eluant is drawn from the solvent reservoirs, the eluant
composition being determined by the proportion of each solvent
delivered to the column via a high-pressure pump and a solvent
mixing system
• Sample mixture is applied to the top of the column and the
components of the mixture are carried down through the column by
the eluant at a rate which is inversely proportional to the attraction
for the packing material
• Passage of the solute from the column is monitored by the detector
and the response is displayed on either a chat recorder or an
integrator

Chaitali S P Tendulkar, PES RTBCOP 11

• Resolution of sample mixtures effected by using a solvent of
constant composition (isocratic) or changing the eluant
composition in a controlled manner during the development
of the chromatogram (gradient elution techniques)
• Incase of low pressure mixing procedure solvents A and B
are mixed on the low pressure side of the pump and vice
versa in high pressure pump

Chaitali S P Tendulkar, PES RTBCOP 12

1. Reservoir holds the solvent
2. A high-pressure pump is used to generate and meter as specified flow
rate of mobile phase, typically few ML per minute
3. An injector is able to introduce the sample into the continuously
flowing mobile phase stream that carries the sample to the HPLC
4. The column contains the chromatographic packing material needed to
carry out separation
5. This packing material is stationary phase because it is held in place by
the column hardware
6. A detector is needed to see the separated compounds eluted from the
HPLC column
7. The mobile phase exits the detector and can be sent to waste or
collected as desired
Chaitali S P Tendulkar, PES RTBCOP 13
Chaitali S P Tendulkar, PES RTBCOP 14
SOLVENT RESERVOIR AND SOLVENT
TREATMENT SYSTEM
• May be made up of glass or Stainless steel and each may contain 200-
1000ml of solvent
• Simplest reservoir- glass bottle with the cap drilled to take PTFE tube to
carry MP from the reservoir to the pump
• Liquid entering the pump should not contain any dust or other particulate
matter
☒Interfere with pumping action
☒Cause damage it gets into seals and valves
☒Can collect at the top of the column causing irregular behavior or blockage
ØFitted with stainless steel filter element that is a push fit on the end of the
tube in the reservoir or in-lineChaitali
filterS P Tendulkar, PES RTBCOP 15
Chaitali S P Tendulkar, PES RTBCOP 16
Degassing
• Important to remove dissolved gases or suspended air bubbles
• HPLC- Equipped with degasser to remove dissolved air and other gases
like O2, N2 which interfere by forming air bubbles in column and affect
detector performance
• Degasser may be a vacuum pumping system or system for sparging in
which dissolved gases are swept out of solution by fine bubbles of inert
gas of low solubility like He
• This degassing system can be inbuilt or can be done separately by
pretreatment
• Eg. Filtering through Millipore filter under vacuum. This removes gases as
well as suspended particulate matter
Chaitali S P Tendulkar, PES RTBCOP 17
Chaitali S P Tendulkar, PES RTBCOP 18
Gradient system
• Different solvents can be used to enhance separation. Most
systems employ 2-3 solvents that differ in the polarity
• The ratio of these solvents is changed in a programmed way
• HPLC has devices to introduce solvents from 2 to 3 reservoirs
into mixing chamber at rates that vary continuously
• Solvent delivery modules are required to deliver a pulse free
flow of eluant to the column at flow rates ranging from 0.1
to 10ml/min

Chaitali S P Tendulkar, PES RTBCOP 19

SOLVENT DELIVERY-PUMPING SYSTEM
• Particle size used in HPLC column 3 to 10µm
• When packed into column- small size of particles leads to a
considerable resistance to solvent flow (back pressure ) – mobile
phase has to be pumped through the column under high pressure
• Pump is positioned in the upper stream of the liquid chromatography
system and generates flow of eluant from the solvent reservoir into
the system

Chaitali S P Tendulkar, PES RTBCOP 20

Criteria for a suitable pump can be
summarized as follows:
1. Material of construction should be inert towards the solvent used
2. Pump should be capable of delivering high volumes of solvent
3. Should be capable of delivering precise and accurate flow
4. Pump head should have a small volume to facilitate rapid changeof
solvent composition
5. Should be capable of delivering high pressures up to around
7000psi
6. Should deliver a pulse free flow and hence not contribute to
detector noise

Chaitali S P Tendulkar, PES RTBCOP 21

 Three
main •Reciprocating
designs
of solvent
•Displacement
delivery •Pneumatic
system
Chaitali S P Tendulkar, PES RTBCOP 22
Constant pressure or pneumatic pump
• Deliver solvents via a small headed
piston which is driven by a
pneumatic amplifier
• The gas piston is attached to a
hydraulic piston that has a smaller
surface area which pushes the eluant
through the column
• Pressure amplification is achieved in
direct ratio to the piston areas
• Pressure applied to the liquid= gas
pressure x area of gas piston/ area of
hydraulic piston
Chaitali S P Tendulkar, PES RTBCOP 23
Constant pressure or pneumatic pump
• Thus for low inlet pressures (approx. 100psi) it is possible to
obtain large outlet pressures (10,000 psi)
• ADVANTAGES:
1. Low cost
2. Ability to deliver high pressure
3. Stability of flow during the delivery

Chaitali S P Tendulkar, PES RTBCOP 24

Constant pressure or pneumatic pump
• DISADVANTAGES:
1. Gives constant pressure but not constant flow
2. partial column blockage or temperature– lead to poor precision and
accuracy of analysis
3. flow rate dependent on solvent viscosity and column back pressure
4. Chamber has to be refilled with solvent periodically with solvent–
interruption of analysis
5. Design makes it difficult to change solvent compositions
• Still used for slurry packing of columns due to their ability to deliver
high flows and pressure
Chaitali S P Tendulkar, PES RTBCOP 25
DISPLACEMENT OR SYRINGE PUMP
• Piston delivering the solvent is driven
by a digital stepping motor
• Flow delivered is determined by
incremental rotational rate of the
motor
• Piston driven at constant speed during
delivery stroke
• Hence delivers constant flow rate to
the column
üIdeal for reproducibility of retention
time data
⨯Lacks suitable refill mechanism
Chaitali S P Tendulkar, PES RTBCOP 26
RECIPROCATING PUMPS

Chaitali S P Tendulkar, PES RTBCOP 27

Single piston

Chaitali S P Tendulkar, PES RTBCOP 28

Chaitali S P Tendulkar, PES RTBCOP 29
Chaitali S P Tendulkar, PES RTBCOP 30
Chaitali S P Tendulkar, PES RTBCOP 31
Chaitali S P Tendulkar, PES RTBCOP 32
Working:
• Piston is driven in and out from the solvent chamber by an eccentric
cam or gear
• On the forward stroke, the inlet check valve closes and the outlet
check valve opens and the mobile phase is pumped to the column
• On the return stroke, outlet valve closes and the chamber is refilled.
• Single headed reciprocating pump– the mobile phase is being
delivered to the column for half the time that the pump is in
operation, and during the drive stroke of the piston the flow rate is
not constant.

Chaitali S P Tendulkar, PES RTBCOP 33

TWIN PISTON reciprocating pumps
• Piston driven either via a single cam or more commonly by individual
cams on a single rotating drive shaft, by a variable speed motor,
approx. 180ºC out of phase
• As piston A completes its compression stroke, piston B begins its refill
cycle
• Adv: provides constant flow which is easy to control, entirely pulse
free and with a small pump head delivery volume– rapid change of
solvent composition– readily adaptable to gradient elution

Chaitali S P Tendulkar, PES RTBCOP 34

• Triple headed pumps ---the pistons are effectively working 120º out
of phase , with two heads in two different stages of filling as the third
is pumping – constant and smooth flow
Chaitali S P Tendulkar, PES RTBCOP 35
Advantages of Reciprocating pump over other
designs:
• Can be used continuously
• Can be readily made pulse free
• Low piston chamber volume facilitates rapid change in solvent
composition
• Most used because of reproducible retention time data

Chaitali S P Tendulkar, PES RTBCOP 36

Equipment for gradient elution:
Low pressure mixing

High pressure mixing

Quaternary gradient systems

Chaitali S P Tendulkar, PES RTBCOP 37
Low pressure mixing

• In this system the solvent is drawn from the reservoirs at a ratio

determined by the switching valve rate and then passed through a
mixing chamber prior to delivery to the piston head
• The switching one is a micro computer controlled and gradient profile
data is entered via the keyboard for construction of the desired
gradient

Chaitali S P Tendulkar, PES RTBCOP 38

High pressure
mixing

• High-pressure mixing uses two pumps whose flows are controlled by

a micro computer
• The total flow required for the application is selected and then the
composition of A and B entered
• Eg 75% A– a will flow at 0.75ml/min
• 25% B- will flow at 0.25ml/min
• The solvent streams are then mixed between the pump and the
column

Chaitali S P Tendulkar, PES RTBCOP 39

Quaternary gradient systems
• Pump consists of two pistons, but solvent is delivered to the column
only from one piston, the other piston chamber serves as a reservoir
• Pistons are 180° out of phase such that when piston A is delivering
solvent to the column, the piston reservoir B is being replenished with
solvent.
• Mixing of the component solvents occurs inside the pump and
connecting tubing during the transfer of the solvent from the
reservoir piston to the delivery piston

Chaitali S P Tendulkar, PES RTBCOP 40

Mobile phase
• The eluants employed in HPLC may comprise of water, aqueous buffer
solutions, miscible organic solvents such as methanol and
acetonitrile or a mixture of the above
• Mobile phase is the solvent that moves the solute through the
column
• In each HPLC, the mobile phase interacts with both solute and the
stationary phase and has a powerful influence on solute retention
and separation
• The chemical interactions of the mobile phase and sample, with the
column, determine the degree of migration and separation of
components contained in the sample
Chaitali S P Tendulkar, PES RTBCOP 41
Solvents used as MP in HPLC should have
following characteristics:
☞Non corrosive to HPLC system components
☞High purity, dust free and should be filtered and degassed before use
☞If UV detection is being employed, should be transparent to the
wavelength employed
☞Low viscosity, low toxicity and low flammability
• Polarity index ‘P’ serves as a rough guide to the strength of the
solvent

Chaitali S P Tendulkar, PES RTBCOP 42

SAMPLE INTRODUCTION
Valve injection

Syringe injection

Stopped flow
injection
Chaitali S P Tendulkar, PES RTBCOP 43
1. Valve injection
üConsists of sample loop
üPreferred and accepted technique
üSample application is rapid
üThe solvent flow from the pump does not have to be stopped and the
systems are easy to use
üReadily adapted for automatic injections
üCan operate at pressures up to 6000psi with reproducibility > 0.2%
v Six port valves usually fitted with external or internal
sample loop

Chaitali S P Tendulkar, PES RTBCOP 44

• With these devices , sample is first transferred at atmospheric pressure
from a syringe into a sample loop . Turning the valve from load to inject
position connects the sample loop into the high pressure MP stream,
whereby the contents of the sample loop are transferred onto the column

Chaitali S P Tendulkar, PES RTBCOP 45

• Internal loop: Volume of the internal loop formed from a machined groove in the
surface of the rotor
• In load position– the channel is isolated from the solvent flow through the
column and can be filled with the sample (fig a)
• On turning the rotor to the inject position channel containing sample is relocated
at the solvent outlet ports and the sample is flushed onto the column
üMethod is simple to use
üExtra column volume due to connecting tubing is minimized
Disadvantage:
⨯ Internal loops are fixed and can
only be altered by changing the
rotor

Rotor fill Chaitali S P Tendulkar,

SamplePES RTBCOP
inject 46
• External interchangeable loop valves marketed by Valco and Rheodyne allow
ready exchange for desired sample size 5, 10, 20 and 50µl
• Operation of valves involve load and inject position
• Microsample injection valves capable of delivering 0.5- 5µl are also available

Chaitali S P Tendulkar, PES RTBCOP 47

2. Syringe injection
• Involves injection of sample through self sealing septum
• Septum is coated with PTFE to prevent attack by organic
solvents
• Greatest column efficiency is obtained by application of
sample via syringe directly onto column bed as:
üBack diffusion of sample is minimized
üApplication as small point source results in transport of
component bands through core of column well away from
walls
üInexpensive
⨯Cannot be used at high back pressure associated with
modern packing materials; historical interest
Chaitali S P Tendulkar, PES RTBCOP 48
• Syringe injection are of two types:
1. Single septum
2. Double septum
• Single septum is used for pressure of approx. 1500psi
• Double septum is used for pressure of approx. 3000psi

Chaitali S P Tendulkar, PES RTBCOP 49

3. Stop flow injection (on-column)
• The pump is stopped and isolated by three way valve from the
column, the sample is then loaded via a syringe through an injection
port which does not contain a septum
• The pump is then re-started, the flow is restored by switching the
valve and the sample is rapidly flushed onto the column

Chaitali S P Tendulkar, PES RTBCOP 50

Autosampler

Chaitali S P Tendulkar, PES RTBCOP 51

Chaitali S P Tendulkar, PES RTBCOP 52
Autosampler

Chaitali S P Tendulkar, PES RTBCOP 53

COLUMNS IN HPLC

Chaitali S P Tendulkar, PES RTBCOP 54

COLUMN, COLUMN PACKINGS , STATIONARY
PHASE
vThe column is most commonly used or made of 316 grade stainless
steel to cope with the high back pressure and are glass lined to
prevent metal catalysis of the solvent
vInside of the stainless steel tube should be as smooth as possible
vCommon dimensions are:
Internal diameter – 4.6mm
External diameter- 6.35mm and up to 25cm long
vMost manufacturers offer a range of lengths and diameter eg.
Lengths of 10, 12.5, 15 or 25 cm and internal diameter of 3, 4.6, 6.2
or 9mm
Chaitali S P Tendulkar, PES RTBCOP 55
Column packing
Material used to pack the column
are of two types:
1. Porous particle and
superficially porous particle
2. Totally porous
• Totally porous microparticulate- 3,5,10 microns
• Superficially porous or micropellicular- consists of a layer of porous
stationary phase on solid core usually a glass bead
• Because of the size, if the core the particle size is relatively larger; mainly
used as the support in conditioning precolumn or guard column
Chaitali S P Tendulkar, PES RTBCOP 56
Column packings
• Packings- Microporous particles of varying size, shape, porosity
• The surface of these particles ---modified by either physical or
chemical means to afford access to any of the classical modes of
chromatography.
• Most common- Silica-- withstand the relatively high pressures in use
and is available with large surface area (200-300 m2g-1) and small
particle size.
• Other microporous packing materials based on alumina, zirconium
and ion exchange materials are also in use.

Chaitali S P Tendulkar, PES RTBCOP 57

• Microporous particles (3-10 µm) give columns that are as much
as 20 times as efficient as porous layer-bead or pellicular (40
um) packings.

Chaitali S P Tendulkar, PES RTBCOP 58

Column
packings in
HPLC

Chaitali S P Tendulkar, PES RTBCOP 59

Totally porous beads
• Initially packing materials were based on porous beads
• Usually silica or alumina, of 20-40µm particle size;
• Gave poor column efficiencies as the particles were totally
porous and had large volumes of associated stagnant mobile
phase.

Chaitali S P Tendulkar, PES RTBCOP 60

Pellicular packing
• Pellicular packings also known as porous layer beads or
controlled surface porosity supports (that is, solid impervious
beads of high mechanical strength) have the impervious core
constructed from materials such as stainless steel, glass, silica,
alumina or ion exchange resins.
• The chromatographically active layer on the surface is
commonly silica, alumina, an ion exchange material or a
cellulose derivative.

Chaitali S P Tendulkar, PES RTBCOP 61

Pellicular packing
• Advantage:
• Solid impervious core -- did not swell when wetted and could
withstand high inlet pressures without deformation.
• Shallow pore structure– no stagnant layer of MP--improvement in
column performance
• Disadvantage:
• Low surface area associated with shallow pores– limits sample
capacity

Chaitali S P Tendulkar, PES RTBCOP 62

Microporous particles
• Packings based on these particles give a 10-fold increase in
column performance thus allowing shorter columns to be used
consequently giving faster separations and requiring smaller
operating pressures.
• The surface of these particles can subsequently be modified by
either physical or chemical means to afford access to any of the
classical modes of chromatography.

Chaitali S P Tendulkar, PES RTBCOP 63

Microporous particles
• The use of microporous particles of 3, 5 and 10 µm
• Advantage:
• Because of the small particle size stagnant mobile phase mass
transfer effects are minimised.
• The large surface area with the concomitant increase in sample
capacity.
• The totally porous microparticle is generally a high surface area,
small porosity silica gel or alumina.

Chaitali S P Tendulkar, PES RTBCOP 64

• Angular silicas such as Partisil and Lichrosorb are prepared by
the reaction of sodium silicate with hydrochloric acid. Spherical
particles
• e.g.Spherisorb are synthesised by the hydrolytic
polycondensation of poly-ethoxysilane followed by emulsion
precipitation or spray-drying.

Chaitali S P Tendulkar, PES RTBCOP 65

• In choosing a column the polarity of SP is matched roughly with that of
the analyte
• A mobile phase of considerably different polarity is then used for elution
• Column selection is usually based upon the sample type, molecular weight
and its applications

Chaitali S P Tendulkar, PES RTBCOP 66

Bonded phase/ Chemically modified silica
supports
• Silica has silanol (-OH) groups which are chemically modified so as to
alter the properties of the silica surface
• Most important is non polar C-18 type
• Different C-18 packing from different manufacturers differs by:
§ Size and shape
§ Pore size of the silica particles
§ Carbon content (% coverage)
§ Extent of end capping

Chaitali S P Tendulkar, PES RTBCOP 67

Bonded phase in HPLC
• Solvent stripping of the stationary phase from the analytical
column and the incompatibility with gradient elution techniques,
led to the development of a range of chemically bonded
stationary phases almost all of which are based on 3, 5 and 10
µm silicas.

Chaitali S P Tendulkar, PES RTBCOP 68

Why chemically bonded??
• Polar, non-ionic, ionic and ionisable molecules can be effectively
separated using a single column and mobile phase
• Stationary phases of a wide range of polarities are available
• It permits a wider choice of eluant
• The predominant eluant is water-based with organic modifiers such
as methanol and acetonitrile which are all readily available and
inexpensive
• Gradient elution techniques can be used without fear of stripping the
stationary phase
• The systems come to equilibrium much faster with little evidence of
irreversible sorption and tailing
• There is no restriction on solvent inlet pressure.

Chaitali S P Tendulkar, PES RTBCOP 69

Bonded phases available may be
classified as follows:
• hydrocarbon groups, such as octadecyl (C18H37), but also
groups with shorter chain lengths such as C1, C2, C8 and aryl;
• polar groups such as amino and cyanopropyl, ethers and diols;
and
• ion exchange groups such as sulphonic acid, amino and
quaternary ammonium.

Chaitali S P Tendulkar, PES RTBCOP 70

Reactions for bonded phase silica

Chaitali S P Tendulkar, PES RTBCOP 71

• The concentration of unreacted
silanols in non-polar bonded phases is
reduced by process known as end
capping in which most of the
remaining silanols are reacted with a
small silylating agent, such as
trimethylchlorosilane
• Figure: surface structure of
octadecylsilane bonded C-18 alkyl
groups and a small number of free
silanols

Chaitali S P Tendulkar, PES RTBCOP 72

Chaitali S P Tendulkar, PES RTBCOP 73
Partition chromatography (HPLC)
• Liquid Liquid C systems consist of a mobile phase and a stationary
phase which is held on the support by phys-ical forces of adsorption.
The column materials are commonly prepared using the solvent
evaporation technique developed for GC.
• In order to avoid solvent stripping of the stationary phase, it is
necessary for the eluant to be saturated with the stationary phase
component.
• The mobile and stationary phases should have contrasting polarities
and be immiscible.

Chaitali S P Tendulkar, PES RTBCOP 74

Partition chromatography (HPLC)
• LLC systems can be classified as either normal or reverse phase.
• In reverse phase partition HPLC, the relative polarities of the
stationary and mobile phases are the opposite to those in normal
phase. The stationary phase is less polar than mobile phase.
• The stationary phases include silica, chemically bonded through
siloxane (Si-O-Si-C) linkage to a low polar functional group.

R= C6H13 (hexyl), C8H17 (Octyl),

C18H37 (octadecyl)
Chaitali S P Tendulkar, PES RTBCOP 75
GUARD COLUMN OR PRE COLUMN
• A short guard column maybe introduced before the
analytical column to increase life of analytical column
üRemoves particulate matter and contaminants from
solvents
üTrap sample components which bind irreversibly to SP
üIn partition chromatography, so as to saturate the MP with
liquid SP so that losses of the solvent from analytical
column are minimized
• Composition similar to analytical column; particle size
usually larger to minimize pressure drop
• On contamination, guard column is replaced; sacrificed to
protect the more expensive analytical column
Chaitali S P Tendulkar, PES RTBCOP 76
Guard column

Chaitali S P Tendulkar, PES RTBCOP 77

DETECTORS:
• The function of the detector in HPLC is to monitor the column eluent
and offer a means of detecting solutes therein.
• The output of the detector is an electrical signal that is proportional
to some property of the mobile phase and/or the solutes
• The detector response will be related to the amount of analyte in the
column effluent.
• Various detector designs have been used and these may be classified
as those :
• which monitor a specific property of the solute or
• those which detect changes in a bulk property of the column effluent

Chaitali S P Tendulkar, PES RTBCOP 78

Examples
• Specific property: Ultraviolet spectrophotometer which may be of a
fixed wavelength (usually 254 or 280nm) or variable wavelength
design. The detector functions by monitoring the changes in
absorbance as the solute passes through the detector flow cell i.e. it
utilizes the specific property of the solute to absorb ultraviolet
radiation
• Bulk property: Refractive index monitor which functions by recording
the refractive changes in the eluant as the solute passes through the
detector cell
• Bulk property detectors though more versatile are generally several
orders of magnitude less sensitive than specific property detectors

Chaitali S P Tendulkar, PES RTBCOP 79

Properties of an ideal HPLC detector :
1. High sensitivity
2. Good stability and reproducibility
3. Linear response over several orders of magnitude
4. Small internal volume minimizing zone broadening
5. A short response time independent of flow rate
6. Insensitive to changes in temperature and pressure
7. High reliability and ease of use
8. Similar response to analytes or selective response to analyte classes
9. Non-destructive

Chaitali S P Tendulkar, PES RTBCOP 80

Chaitali S P Tendulkar, PES RTBCOP 81
Bulk property detectors Detectors which function by
• Refractive index desolvating: separating the
solvent from the eluant thus
• Density
allowing subsequent detection
• Dielectric constant Eg. FID or mass spectrometry
• Electrochemical
Pre or post column
Solute property detectors derivatization: involving
chemical reaction of the
• UV Visible analyte
• Fluorescence
• Diode array
• Mass spectrometry
Chaitali S P Tendulkar, PES RTBCOP 82
UV-VISIBLE DETECTORS
• UV visible detectors are most used detectors due to its specific
response to the class of compounds or particular compounds
depending on the functional groups of eluting molecules that absorb
light, although some compounds with no light absorbing groups give
suitable response after post column derivatisation to introduce light
absorbing entities
• Function by monitoring the light absorbed by the solute molecules
from the incident beam
• They are not appreciably flow or temperature sensitive, have a good
dynamic linear range but are however selective

Chaitali S P Tendulkar, PES RTBCOP 83

• The major modification required to adapt conventional spectrometers for use
in HPLC is to design suitable flow-cells.
• The volume of a well resolved peak in HPLC can be as little as 150 µl and
considerably smaller for micro-bore, and thus a small volume flow-cell which
will avoid excessive band broadening and which is capable of withstanding
pressures of several bar is required.
• Typically, the flow-cell for spectrophotometric detection in HPLC has a
pathlength of 10 mm and a bore of 1mm giving a volume of 8µl.

Chaitali S P Tendulkar, PES RTBCOP 84

Three common types of UV visible detectors
1. Fixed wavelength detectors
2. Variable wavelength detectors
3. Photo diode array detectors

• FIXED WAVELENGTH DETECTOR: absorbance of only one given wavelength

is monitored by the system (usually 254 nm or 280 nm)
💡Mercury lamp emits at 254nm (organic compounds containing
conjugated chromophores, aromatic ring or heterocyclic rings)
💡Mercury lamp + phosphor emit at 280nm
üSimplest and cheapest of the UV detectors
üLimited in flexibility
üLimited in types of compounds that can be monitored

Chaitali S P Tendulkar, PES RTBCOP 85

• Variable wavelength
detector
• A single wavelength is monitored
at any given time but any
wavelength in the wide spectrum
range can be selected
• Wavelength can vary from 190 to
800 nm
• To increase the specificity and
sensitivity the Hg has been
replaced by
💡Deuterium (190 to 400nm)
💡Tungsten (400 to 700nm)
Coupled with manually adjustable
diffraction grating monochromator
Chaitali S P Tendulkar, PES RTBCOP 86
💰More expensive, requires more advanced optics
üMore versatile, used for wider range of compounds
üMore sensitive due to photo multiplier tube or
amplification circuit

Chaitali S P Tendulkar, PES RTBCOP 87

Chaitali S P Tendulkar, PES RTBCOP 88
 Photo diode array technology allows
PHOTODIODE ARRAYS continuous scanning of the
absorbance spectrum of the
chromatographic eluant, thus
eliminating the need to stop the flow
of the mobile phase

Chaitali S P Tendulkar, PES RTBCOP 89

PHOTODIODE ARRAYS
• Reversed optic geometry is used; operates by simultaneously monitoring
absorbance of solutes at several different wavelengths
• All of the light from broad emission source is focused on to the sample
• Full spectrum of light emerging from flow cell dispersed by holographic
diffraction grating into single wavelength which are focused simultaneously
on linear array of photo diode detectors
• When transmitted radiation falls on the photo diode, currents are
generated
• Electronic signals from each photo diode detector is processed to give
absorbance data displayed as spectrum across the complete spectral range
• Data can be displayed as function of wavelength and time
• Spectra obtained from diode array spectrometers are useful in component
identification

Chaitali S P Tendulkar, PES RTBCOP 90

Chaitali S P Tendulkar, PES RTBCOP 91
Fluorescence detector
• A small number of compounds possess the ability to fluoresce
• When compounds having specific functional groups are excited by shorter
wavelength energy and emit higher wavelength radiation- fluorescence
• Usually, the emission is measured at right angles to the excitation
• Can be used with gradient elution system
• Fluorescence-based applications like quinoline, steroids, alkaloids etc
• Also for samples which can be made fluorogenic by reacting with
fluorogenic reagents example ninhydrin for amino acids
• Fluorescence intensity depends on both the excitation and emission
wavelength, allowing selectively detect some components which suppress
is the emission of others
Chaitali S P Tendulkar, PES RTBCOP 92
Chaitali S P Tendulkar, PES RTBCOP 93
Refractive index detector
• Bulk property detector
• These detectors sense the difference in refractive index between the
column eluant and a reference stream of pure mobile phase.
• Low sensitivity compared to UV detector
• Difficulties arise due to the sensitivity of the solvent RI to fluctuations in
temperature, pressure and presence of dissolved gases and eluant
composition.
• Three types of commercially available RI detectors:
ØDeflection
ØFresnal
ØInterference

Chaitali S P Tendulkar, PES RTBCOP 94

• For dilute solutions: Additivity law of refractive index is applicable
• Nc= (N1V1+ N2V2)/ (V1+ V2)
Nc- composite RI of the solution in flow cell
N1 and N2- are RI of pure solvent and solute
V1 and V2 volume of each present

• Suitable for detecting all components

(samples which do not have UV
absorption, such as sugar, alcohol or
inorganic ions) as change in refractive
index occurs for all analytes.

Chaitali S P Tendulkar, PES RTBCOP 95

Fresnal refractometer
• Based on Fresnel's Law, which states
that the fraction of light reflected at a
glass-liquid interface varies with the
angle of incidence and the RI of the
liquid.
• The beams produced by the source and
collimating lens are focused onto the
reference and sample prism-liquid
interfaces.
• The light is then refracted through the
liquid in the cell, reflected off the
backing surface and then passed
through the liquid-prism interface on to
the detection system.

Chaitali S P Tendulkar, PES RTBCOP 96

• The beams produced by the source and collimating lens are focused
onto the reference and sample prism-liquid interfaces. The light is
then refracted through the liquid in the cell, reflected off the backing
surface and then passed through the liquid-prism interface on to the
detection system.
• With mobile phase in both cells the beams are refracted equally and
light beams of equal energy fall on the dual element photodetector.
With analyte in the sample cell the light beams are refracted to
differing extents and different amounts of radiation fall on the
photodetector.
• The disadvantages of this system are (a) two prisms are required to
cover the range of RI of solvent encountered (b) small volume cells
(5 pl) help enhance sensitivity; however the limited sensitivity (×1000
less than ultraviolet) and stability are the principle limits to the
universal application of this technique.

Chaitali S P Tendulkar, PES RTBCOP 97

 Deflection refractometer
• In the deflection refractometer light from
the source is collimated by the lens and
falls on the detector cell, which consists of
sample and reference sections that are
separated by a dividing plate
• As the incident light passes through the
cell it is refracted, reflected and refracted
again, before passing through the lens
and on to the photodetector.
• The signal produced is proportional to the
position at which the beam strikes the
detector which is dependent upon the RI
within the sample compartment

Chaitali S P Tendulkar, PES RTBCOP 98

 Shearing refractometer

• In a shearing interferometer radiation of known wavelength (546

nm) is split into two beams of equal intensity by the beam splitter.
These beams are then focused by the lenses and pass through
the reference and sample cells

Chaitali S P Tendulkar, PES RTBCOP 99

• The beams are then focused onto a second beam splitter, B,
which corrects the wavefronts so that the beams are in phase
and interfere.
• When solvent is present in both sample and reference cells
constructive interference occurs. However, if solute is present
in the sample compartment, the sample beam has a different
optical path length, which can lead to destructive interference
when the sample and reference beams interact after beam
splitter B as the beams are now out of phase.

Chaitali S P Tendulkar, PES RTBCOP 100

Mass spectrometer
• Analytes are detected based on molecular fragmentation and separation
based on the mass to charge ratio of fragmented species
• Obtained information especially useful for structure identification.
However, its use is not limited to structure identification and can be used
to quantify very low detection limit of elemental and molecular
components
• Detector have various mass analyser depending upon that application.
Some of the analysers are quadrapoles, ion trap, time of flight
• Detectors provide very high sensitivity selectivity and time resolution
• Method combines separation power of HPLC with detection power of mass
spectrometry

Chaitali S P Tendulkar, PES RTBCOP 101

Chaitali S P Tendulkar, PES RTBCOP 102
Electrochemical detector
• Measure either the conductance of the eluent or the current
associated with the oxidation or reduction of solute.
• Conductivity detectors are used for the detection of inorganic or
organic ions which are electroactive
• A substance which can be electrochemically oxidized or reduced is
known as electroactive
• Electrochemical detectors which deal with the measurement of
current associated with the oxidation or reduction of solutes are
called amperometric or coulometric detectors.

Chaitali S P Tendulkar, PES RTBCOP 103

 Electro reducible: Aromatic amines,
phenols, carbohydrates, oximes, alcohols

Electro-oxidizable: Ketones, activated

halogens, nitrates, aldehydes

• Consists of working electrode, reference electrode (Ag/AgCl or Pt)

• A pre selected potential is applied across electrodes.
• When current is passed through a solution, reactions occur at each electrode in which
electron exchange takes place between the electrode and substances in solution.
• At the cathode, substances gain electrons (reduction) and at anode they lose electrons
(oxidation)
• As electrochemically species passes through cell it is oxidized and reduced; current
generated in flow cell is continuallyChaitali
monitored.
S P Tendulkar, PES RTBCOP 104
Radiochemical detector
• Monitor radiolabelled compounds as they elute from the LC column
• Radioactive compounds eluded from the column are detected using
gas field detectors such as Geiger Muller counter, scintillation
detectors and solid state detectors like NaI
• Common labels 14C, 3H, 32P, and 35S are low energy beta emitters and
hence sensitivity of these detectors is low
• Improvement achieved by– either by increasing volume of flow cell or
decrease flowrate so as to increase the residence time in the flow
cell.

Chaitali S P Tendulkar, PES RTBCOP 105

Chaitali S P Tendulkar, PES RTBCOP 106
RECORDER
• The change in eluant detected by a detector in the form of an
electronic signal – is not visible to the eyes
• In older days– the pen (paper) chart recorder was popularly used
• Nowadays- a computer based data processor( integrator) is more
common
• Various types of data processors range from simple system consisting
of inbuilt printers and word processor while those with software that
are specifically designed for a LC--- allow data acquisition with
features like peak fitting, baseline correction, automatic
concentration calculation, molecular weight determination
Chaitali S P Tendulkar, PES RTBCOP 107
APPLICATIONS OF HPLC
• QUANTITATIVE ANALYSIS
• Involves the measurement of peak height or peak area with that of
standard
• For paks that are well resolved, both peak height and area are proportional
to the concentration
vAnalysis based on peak height
• Obtained by connecting the baseline on either side of peak by straight line
and measuring perpendicular distance from the line to the peak
• Accurate results obtained if variation in column conditions do not alter
peak with during the time chromatogram is obtained for sample and
standard
• Important to control column temperature, eluant flow and rate of sample
injection; also avoid overloading column.

Chaitali S P Tendulkar, PES RTBCOP 108

vAnalysis based on peak area
ØMost modern instruments have Digital electronic integrators which
permit precise estimation of peak areas
ØManually, for symmetrical peaks, area obtained by product of peak
height with width at one half of peak height
ØOr by cutting out peak and determining its weight a relative to weight
of known-area of recorder paper
ØIn case of single point standardization: solution of reference standard
of known concentration is injected and its peak area obtained.
ØFrom area of unknown its concentration can be calculated by using
the formula
• Conc Unknown = Area unkown X Conc known
Area known

Chaitali S P Tendulkar, PES RTBCOP 109

vCalibration and standards
• Here calibration plot of peak area/peak height of standard solutions
of known concentration versus concentration of solution is plotted.
• Plot should yield straight line passing through origin and from peak
area or height of unknown, its concentration can be estimated
• Two methods are used external standard and internal standard

Chaitali S P Tendulkar, PES RTBCOP 110

• EXTERNAL STANDARD
• Accuracy of this method is dependent upon the reproducibility of the
injection volume
• In multiple point standardization, standard solution of known
concentrations of the compound of interest are prepared covering the
range of unknown concentration
• Sample solution is injected
• Peak height or area is then plotted versus the concentration for each
standard
• The plot should be linear and pass through origin
• The concentration of the unknown is then determined from the
calibration curve

Chaitali S P Tendulkar, PES RTBCOP 111

Chaitali S P Tendulkar, PES RTBCOP 112
• Internal standard method
• In this method an equal amount of an internal standard , a
component that is not present in the sample, is added to both the
sample and standard solutions
• Quantification is achieved by using ratios of peak height or area of the
component to the internal standard
• Conc soln= Area internal std in knownX Area unknown X Conc known
Area internal std unknown X Area known
Internal standard selected should be chemically similar to, have similar
retention time and derivatize similarly to the analyte, should be stable
and not interfere with any of the sample components
Internal standard should be added before any preparation of the
sample

Chaitali S P Tendulkar, PES RTBCOP 113

Chaitali S P Tendulkar, PES RTBCOP 114
• Area Normalization method
• For quantitative analysis we assume that the area (or the height) of
our peak in the chromatogram is proportional to the amount of
substance that produced the peak
• In the simplest method we measure areas or heights which are then
normalized (this means that each area or height is expressed as a
percentage of the total). The normalized heights or areas give the
composition of our mixture, as in the following example
Peak number Peak height Normalized peak height=
% w/w
1 12 12/162 * 100= 7.4
2 27 16.7
3 72 44.4
4 51 31.5
Total= 162 100
Chaitali S P Tendulkar, PES RTBCOP 115
Chaitali S P Tendulkar, PES RTBCOP 116
DERIVATIZATION IN HPLC
• Chemical reaction between an analyte and a reagent to change the
chemical and physical properties of analyte
• Derivatization reaction should be rapid, quantitative and reduce
minimal by products
• Excess of reagent should not interfere with analysis or be easily
removed from reaction matrix
vWHY???
• Some compounds cannot be detected or detection limits maybe
sufficiently low for practical application
• Problem can be solved by the derivatizing analyte to a new compound
or complex that will have sufficient response to conventional detectors
Chaitali S P Tendulkar, PES RTBCOP 117
Reasons:
• To allow chromatography of compound that otherwise could not be
detected by the detector currently available eg. aliphatic amines,
alcohols, carboxylic acid
• To improve resolution by functional group modification – this
enhances the interaction of solute is with stationary phase eg.
esterification of acid.
• To improve sensitivity of detection eg. Formation of fluorescent
derivatives of amino acid
• To improve stability of analyte

Chaitali S P Tendulkar, PES RTBCOP 118

Sample derivatisation
• Derivatization of a sample is undertaken principally for two reasons:
• First, there is no detector for HPLC that has universally high sensitivity for all
solutes; hence, the use of a suitable chemical transformation of the solute can
greatly extend the sensitivity and versatility of a selective detector.
• Second, sample derivatization may be undertaken to enhance the detector
response to sample bands relative to overlapping bands of no analytical interest.
• These reactions can be carried out either before or after the passage of solute
through the column.
• The reagents are classed as either fluoregenic, i.e. non-fluorescent molecules
which react with analytes to form fluorescent derivatives, or chromogenic
reagents which react with the analyte to yield a derivative that strongly adsorbs
ultraviolet or visible radiation.

Chaitali S P Tendulkar, PES RTBCOP 119

PRE COLUMN DERIVATIZATION
• Before sample is introduced in the column
• Sample is derivatized and introduced in the HPLC column
• Provides functional group which enhances the separation of solutes
as well as their detectability
• Fluoregenic reagents: eg. o-Pthalic anhydride (OPA), Fluorescamine,
Napthyl Isocynate
• Chromogenic eg. Derivatisation of carboxylic acid with
napthylbromide ; formation of pthaldehyde derivatives of amino acids

Chaitali S P Tendulkar, PES RTBCOP 120

PRE COLUMN DERIVATIZATION
• An example of pre-column derivatisation, to enhance the
spectrophoto- metric response of the sample, is the reaction between
naphacyl bromide, the derivatising agent, and carboxylic acids.
• The ester product contains a chromophore unit and the sample
derivatives are now able to absorb ultraviolet radiation

Chaitali S P Tendulkar, PES RTBCOP 121

PRE COLUMN DERIVATISATION
• Pre column derivatization can be carried out manually, off-line from
the system and places little restriction on the choice of reagents,
solvents, length of reaction time, etc., all of which are important
restrictions in post-column applications.
• The reaction, however, may give rise to more than one product which
can subsequently complicate the chromatogram.
• For quantitative work, the extent of the reaction must be precise and
reproducible for different sample concentrations.

Chaitali S P Tendulkar, PES RTBCOP 122

POST COLUMN DERIVATIZATION
• After the sample is eluted and before it reaches the detector
• Allows separation of solute based on their own functionalities but
introduces a reagent into the column effluent before it reaches the
detector in order to increase sensitivity
• Special equipment are available for Post column derivatization that have
capability of adding reagent
ØReaction of amino acids with ninhydrin
ØReaction of amino acids with pthaldehyde as derivatizing agent introduces
a fluorescent group into the molecule
ØReaction of fatty acid with o-nitrophenol
ØReaction of ketones with 2,4-DNP
Chaitali S P Tendulkar, PES RTBCOP 123
POST COLUMN DERIVATIZATION
• An example of post-column derivatisation is the reaction of amino
acids with ninhydrin; again a chromophoric unit is present in the
sample derivatives.
• The replacement of ninhydrin by ortho-phthaldehyde as the
derivatizing agent introduces a fluorescent group into the molecule
and thus enables the highly sensitive fluorescent detector to be
employed.

Chaitali S P Tendulkar, PES RTBCOP 124

POST COLUMN DERIVATIZATION
• Post column derivatization has advantage that it may be readily
incorporated as part of a fully automated system and thus will not
complicate the chromatography even if the reaction yields more than
one product.
• Major consideration à extra-column effects which can impair the
overall resolution of the LC system.
• For post-column reactors it is not essential that the derivatisation is
carried out to completion, only that the extent of reaction is
reproducible.

Chaitali S P Tendulkar, PES RTBCOP 125

TYPES
• For UV-vis spectrophotometric detection
• For Fluorometric detection
• For chiral analysis

Chaitali S P Tendulkar, PES RTBCOP 126

QUESTIONS
• 10 marks (Long answers)
• Describe the instrumentation of HPLC system with the help of a neat
labelled diagram. Add a note on applications in HPLC (Aug 2022)
• Give advantages of as HPLC as separation technique over GC. Explain
the instrumentation in HPLC ( Jan 2023)

Chaitali S P Tendulkar, PES RTBCOP 127

QUESTIONS
• 5 marks ( Short answers)
• Discuss the instrumentation of HPLC system with the help of a neat
labeled diagram ( Feb 2022)
• Discuss

Chaitali S P Tendulkar, PES RTBCOP 128

QUESTIONS
• 2 marks ( Very short answers)
• Write any two pumps used in HPLC ( Feb 2022)
• Write any two stationary phases used in reverse phase HPLC (Feb
2022, July 2023)
• Write detector types used in HPLC ( Aug 2022, Jan 2023)

Chaitali S P Tendulkar, PES RTBCOP 129